Marijuana is the most widespread illegal drug of abuse in Western societies. The main active ingredient of marijuana, Delta9- tetrahydrocannabinol (THC), binds to selective G protein-coupled receptors, called cannabinoid receptors. Activation of these receptors by THC exerts profound behavioral effects in humans. This suggests that endogenous neurotransmitter substances that engage cannabinoid receptors may serve important physiological and pathological functions. Although an endogenous cannabinoid ligand, anandamide, was recently identified, evidence indicates that additional ligands may exist. On the basis of our preliminary results, we propose to test the hypothesis that 2- arachidonylglycerol (2-AG), a product of phospholipid metabolism, constitutes a second endogenous ligand for brain cannabinoid receptors. To test this hypothesis it will be necessary to show that the enzymatic machinery for the formation and inactivation of 2-AG is present in neurons. Moreover, it will be necessary to demonstrate that 2-AG binds to and activates neuronal cannabinoid receptors. The first aim of the proposed research is to investigate the mechanisms responsible for 2-AG production, using rat brain neurons in primary culture. Preliminary experiments have shown that these neurons produce substantial amounts of 2-AG. The proposed studies will identify the transmembrane signalling systems and enzyme pathways underlying 2-AG production. The second aim of our proposed research is to determine whether neural activity is necessary for the formation of 2-AG. Preliminary experiments have demonstrated that, in superfused slices of rat hippocampus, high-frequency electrical stimulation of an identified excitatory pathway (the Schaffer collaterals) results in the marked accumulation of 2-AG. We will examine whether 2-AG formation is stimulated by levels of neural activity that are likely to occur in vivo, and investigate the ion channels and neurotransmitter receptors involved in this response. The third aim of our proposal is to characterize the pharmacological properties of 2-AG in cortical neurons in culture. Initial experiments have indicated that 2-AG activates neuronal cannabinoid receptors. We will determine the potency, efficacy and selectivity of 2-AG in eliciting such response. The fourth aim of the proposed research is to identify the biochemical mechanisms involved in the biological inactivation of 2-AG. Preliminary results suggest that 2-AG is hydrolyzed enzymatically to arachidonate and glycerol, two products that do not activate cannabinoid receptors. We will use subcellular fractions of rat brain tissue to identify and characterize the enzyme activity that mediates 2-AG hydrolysis. In conclusion, by demonstrating that 2-AG meets the necessary criteria of an endogenous cannabinoid ligand, our studies will shed new light on the mechanisms of marijuana abuse and help develop new strategies in the treatment of neurological, psychiatric and substance abuse disorders.